Interleukin-19

ABSTRACT

The present invention concerns a novel human cytokine. In particular, isolated nucleic acid molecules are provided encoding interleukin-19 (IL-19). IL-19 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further concerns therapeutic methods for modulating cytokine production.

[0001] This application claims the benefit of the filing date ofprovisional application No. 60/024,882 filed on Aug. 30, 1996, which isherein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention concerns a novel human cytokine. Inparticular, isolated nucleic acid molecules are provided encodinginterleukin-19 (IL-19). IL-19 polypeptides are also provided, as arevectors, host cells and recombinant methods for producing the same. Theinvention further concerns therapeutic methods for modulating cytokineproduction.

[0004] 2. Related Art

[0005] Interleukin-10 (IL-10) is a pleiotropic cytokine that has beenimplicated as an important regulator of the functions of lymphoid andmyeloid cells. IL-10 blocks activation of cytokine synthesis and severalaccessory functions of macrophages, thus acting as a potent suppressorof the effector functions of macrophages, T cells and NK cells. IL-10has also been implicated in the regulation of differentiation of Bcells, mast cells and thymocytes.

[0006] IL-10 was identified independently in two different lines ofexperiments. One of these identified a B-cell-derived mediator whichco-stimulated active thymocytes (Suda et al., Cell Immunol. 129:228(1990)). The other identification determined that IL-10 is involved inthe cross-regulation between two often mutually exclusive effector armsof immunity carried out by T helper (CD4⁺) subpopulations, Th1 (involvedin cell-mediated immune responses) and Th2 (involved inantibody-mediated immune responses). In this role, IL-10 is expressed byTh2 cells and functions to suppress cytokine production by Th1 cells, anactivity termed cytokine synthesis inhibitory factor (CSIF) activity.

[0007] cDNA clones encoding murine IL-10 (mIL-10) were isolated based onthe expression of CSIF activity (Moore et al., Science 248:1230-34(1990)). cDNA clones encoding human IL-10 (mIL-10) were subsequentlyidentified by cross-hybridization with the mouse cDNA (Vieira et al.,Proc. Natl. Acad. Sci. USA 88:1172-1176 (1991)). mIL-10 is expressed bymouse CD4+Th2 cells, at least one CD8⁺ clone, B lymphomas, T cells,activated mast cell lines, activated macrophages, keratinocytes, andLy-1 B (B-1) cells (Fiorentino, D. F. et al., J. Exp. Med. 170:2081(1989); (Moore et al., Science 248:1230-34 (1990); 87-93 (1992); Lin etal., Ann. N.Y. Acad. Sci. 651 O'Garra et al. Int. Immunol. 2: 821-832(1990); MacNeil et al., J. Immunol. 145: 4167-4173 (1990); Fiorentino etal., J. Immunol. 147:3815-3822 (1991); Hisatsune et al, LymphokineCytokine Res. 11:651-683 (1992)). hIL-10 is expressed by human CD4⁺ Tcells and Th0, Th1, and Th2 T cell clones, by CD8⁺ T cells and clones(Yssel et al., J. Immunol.), monocytes/macrophages, keratinocytes,activated B cells, B lymphomas, and Burkitt lymphoma lines infected witha transforming EBV strain, but not with a non-transforming strain(Vieira, P. et al., Proc. Natl. Acad. Sci. USA 88.1172-76 (1991); deWaal-Malefyt, R. et al., J. Exp. Med. 174.1209-20 (1991);deWaal-Malefyt, R. et al., J. Exp. Med. 174:915-24 (1991); Salgame, P. etal., Science 254:279-82 (1991); Yamamura, M. et al., Science 254:277-79(1991);Ralph, P. et al., J. Immunol. 148:808-14 (1992); Benjamin, D. etal., Blood 80:1289-98 (1992)). Thus, IL-10 is not strictly aTh2-specific cytokine, and its pattern of expression resembles IL-6 morethan IL-4 or IL-5 (Wang, S. C. et al., Transplant. Proc. 23:2920-22(1991)). Like IL-6 but unlike most other T cell derived cytokines, IL-10expression is not inhibited by cyclosporin or FK-506 (Wang, S. C. etal., Transplant Proc. 23:2920 (1991)).

[0008] In an attempt to determine the in vivo role of IL-10, normal micewere treated from birth to adulthood with IL-10-neutralizing antibodies(Wang, S. C. et al., Transplant Proc. 23:2920 (1991); Ishida, H. et al.,J. Exp. Med. 175:1213 (1992)) The resulting phenotypic changes includedan increased level of circulating IFN-γ, TNF-α and IL-6, reduced serumIgM and IgA, a marked depletion of peritoneal B cells, and an inabilityto develop in vivo antibody responses to two bacterial antigens known becombatted with antibody produced by peritoneal B cells (Hayakawa, K. etal., Annu. Rev. Immunol. 6:197 (1988)). The reduction in peritoneal Bcells was determined to be a consequence of IFN-γ elevation (Ishida, H.et al., J. Exp. Med. 175:1213 (1992)).

[0009] Other experiments have shown that IL-10 suppresses in vitroproduction of inflammatory monokines such as TNF-α and IL-1. This datacorresponds to in vivo studies which show that IL-10 antagonists elevatethe same inflammatory monokines. These results predict a stronganti-inflammatory role for IL-10. In addition, IL-10 antagonists may beuseful to enhance Th1 immunity, which could be beneficial in infectiousdiseases of viral origin, or diseases involving intracellular pathogens.

[0010] The diverse biological activities of IL-10 have led topredictions that both IL-10 and its antagonists will have a wide rangeof clinical applications. It is clear that there is a continuing need inthe art for isolating novel cytokines capable of mediating such diversebiological processes.

SUMMARY OF THE INVENTION

[0011] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a human IL-19 polypeptide havingthe amino acid sequence in FIG. 1 [SEQ ID NO: 2] or the amino acidsequence encoded by the cDNA clone deposited in a bacterial host as ATCCDeposit Number 97662 on Jul. 17, 1996. The nucleotide sequencedetermined by sequencing the deposited IL-19 cDNA clone, which is shownin FIG. 1 [SEQ ID NO: 1], contains an open reading frame encoding apolypeptide of about 177 amino acid residues including an initiationcodon at nucleotide positions 44-46, a leader sequence of about 24 aminoacid residues and a deduced molecular weight of about 20.4 kDa. The 153amino acid sequence of the predicted mature IL-19 protein is shown inFIG. 1 (last 153 residues) and in SEQ ID NO:2 (from amino acid residue 1to residue 153).

[0012] The present invention also relates to recombinant vectors whichinclude the isolated nucleic acid molecules of the present invention andto host cells containing the recombinant vectors, as well as to methodsof making such vectors and host cells and for using them for productionof IL-19 polypeptides or peptides by recombinant techniques.

[0013] The invention further provides an isolated IL-19 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0014] IL-19, which is secreted, and which has significant homology toIL-10, is believed by the present inventors to be expressed only, or atleast primarily, in activated monocytes (FIG. 4). Thus, detecting IL-19gene expression in cells of the immune system is useful for identifyingactivated monocytes. Further, for a number of disorders, it is believedby the inventors that significantly higher or lower levels of IL-19 geneexpression can be detected in bodily fluids (e.g., serum, plasma, urine,synovial fluid or spinal fluid) taken from an individual having such adisorder, relative to a “standard” IL-19 gene expression level, i.e.,the IL-19 expression level in bodily fluids from an individual nothaving the disorder. Thus, the invention provides a diagnostic methoduseful during diagnosis of a disorder related to an abnormal level ofIL-19 gene expression, which involves (a) assaying IL-19 gene expressionlevel in cells or body fluid of that individual; (b) comparing thatIL-19 gene expression level with a standard IL-19 gene expression level,whereby an increase or decrease in the assayed IL-19 gene expressionlevel compared to the standard expression level is indicative of adisorder. An additional aspect of the invention is related to a methodfor treating an individual in need of an increased level of IL-19activity in the body, which involves administering to such an individuala composition comprising an IL-19 polypeptide of the invention. A stillfurther aspect of the invention is related to a method of treating anindividual in need of a decreased level of IL-19 activity in the body,which involves administering to such an individual a compositioncomprising an antagonist to IL-19 such as anti-IL-19 antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 shows the nucleotide [SEQ ID NO: 1] and deduced amino acid[SEQ ID NO:2] sequences of the complete IL-19 protein determined bysequencing of the DNA clone contained in ATCC Deposit No. 97662. Theprotein has a leader sequence of about 24 amino acid residues(underlined) and a deduced molecular weight of about 20.4 kDa. The aminoacid sequence of the predicted mature IL-19 protein is also shown inFIG. 1 (last 153 amino acids) [SEQ ID NO:2].

[0016]FIG. 2 is a protein gel showing IL-19 protein expressed from E.coli strain M15rep4 (see Example 1).

[0017]FIG. 3 is a protein gel showing full length and truncated IL-19proteins produced in an in vitro coupled transcription/translationsystem (see Example 3).

[0018]FIG. 4 is a northern blot analysis of IL-19 expression in humantissue (HL-60, THP-1, U937 and primary human monocytes) (see Example 5).

[0019]FIG. 5 shows the regions of similarity between the amino acidsequences of the IL-19 protein and human IL-10 (hIL-10) [SEQ ID NO: 3].

[0020]FIG. 6 shows an analysis of the IL-19 amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 19 to about 28, about 88 to about 106, andabout 139 to about 149 in FIG. 1 correspond to the shown highlyantigenic regions of the IL-19 protein. These highly antigenic fragmentsin FIG. 1 correspond to the following fragments, respectively, in SEQ IDNO:2: amino acid residues about −5 to about 4, about 64 to about 82, andabout 115 to about 125.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the IL-19 protein having the aminoacid sequence shown in FIG. 1 [SEQ ID NO:2] which was determined bysequencing a cloned cDNA. The IL-19 protein of the present inventionshares sequence homology with human IL-10 (FIG. 5) [SEQ ID NO:3]. Thenucleotide sequence shown in FIG. 1 [SEQ ID NO:1] was obtained bysequencing the HMQBM23 cDNA clone encoding an IL-19 polypeptide, whichwas deposited on Jul. 17, 1996 at the American Type Culture Collection,12301 Park Lawn Drive, Rockville, Md. 20852, and given accession number97662. The deposited clone is contained in the pBluescript SK(−) plasmid(Stratagene, La Jolla, Calif.).

Nucleic Acid Molecules

[0022] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain a some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0023] Using the information provided herein, such as the nucleotidesequence in FIG. 1, a nucleic acid molecule of the present inventionencoding an IL-19 polypeptide may be obtained using standard cloning andscreening procedures, such as those for cloning cDNAs using mRNA asstarting material. Illustrative of the invention, the nucleic acidmolecule described in FIG. 1 [SEQ ID NO:1] was discovered in a cDNAlibrary derived from human activated monocytes. The determinednucleotide sequence of the IL-19 cDNA of FIG. 1 contains an open readingframe encoding a protein of about 177 amino acid residues with aninitiation codon at positions 44-46 of the nucleotide sequence shown inFIG. 1 [SEQ ID NO. 1], and a predicted leader sequence of about 24 aminoacid residues, and a deduced molecular weight of about 20.4 kDa. Theamino acid sequence of the predicted mature IL-19 protein is from aboutamino acid residue 25 to about residue 177 shown in FIG. 1 or aminoacids 1-153 shown in SEQ ID NO:2. The IL-19 protein shown in FIG. 1 [SEQID NO:2] is about 20% identical and about 46% similar to human IL-10(FIG. 5).

[0024] The present invention also provides the mature form(s) of theIL-19 protein of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature IL-19 polypeptides having the amino acid sequence encoded bythe cDNA clone contained in the host identified as ATCC Deposit No.97662 and as shown in SEQ ID NO:2. By the mature IL-19 protein havingthe amino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC Deposit 97662 is meant the mature form(s) of theIL-19 protein produced by expression in a mammalian cell (e.g., COScells, as described below) of the complete open reading frame encoded bythe human DNA sequence of the clone contained in the vector in thedeposited host. As indicated below, the mature IL-19 having the aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.97662 may or may not differ from the predicted “mature” IL-19 proteinshown in SEQ ID NO:2 (amino acids from about 1 to about 153) dependingon the accuracy of the predicted cleavage site based on computeranalysis.

[0025] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are available.For instance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) andvon Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0026] In the present case, the predicted amino acid sequence of thecomplete IL-19 polypeptides of the present invention were analyzed by acomputer program (“PSORT”) (K. Nakai and M. Kanehisa, Genomics14:897-911 (1992)), which is an expert system for predicting thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis by the PSORT programpredicted the cleavage site between amino acids −1 and 1 in SEQ ID NO:2.Thereafter, the complete amino acid sequences were further analyzed byvisual inspection, applying a simple form of the (−1,−3) rule of vonHeinje. von Heinje, supra. Thus, the leader sequence for the IL-19protein is predicted to consist of amino acid residues from about −24 toabout −1 in SEQ ID NO:2, while the mature IL-19 protein is predicted toconsist of residues from about 1 to about 153.

[0027] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the actual IL-19 polypeptide encoded by the deposited cDNA comprisesabout 177 amino acids, but may be anywhere in the range of 170-183 aminoacids; and the actual leader sequence of this protein is about 24 aminoacids, but may be anywhere in the range of about 18 to about 29 aminoacids.

[0028] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0029] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0030] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) with an initiationcodon at positions 44-46 of the nucleotide sequence shown in FIG. 1 [SEQID NO:1]; DNA molecules comprising the coding sequence for the matureIL-19 protein shown in FIG. 1 (last 153 amino acids) [SEQ ID NO:2]; andDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode the IL-19 protein. Of course, the genetic code iswell known in the art. Thus, it would be routine for one skilled in theart to generate the degenerate variants described above.

[0031] In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: PO14 (SEQID NO:16).

[0032] Sequences of public ESTs that relate to a portion of SEQ ID NO:1have the following GenBank Accession Numbers: AA151656 (SEQ ID NO:12),AA151652 (SEQ ID NO:13), AA151733 (SEQ ID NO:14), and AA151736 (SEQ IDNO:15).

[0033] In another aspect, the invention provides isolated nucleic acidmolecules encoding the IL-19 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97662 on Jul. 17, 1996. In a further embodiment, nucleicacid molecules are provided encoding the mature IL-19 polypeptide or thefull-length IL-19 polypeptide lacking the N-terminal methionine. Theinvention also provides an isolated nucleic acid molecule having thenucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence ofthe above-described deposited cDNA clone. The invention further providesan isolated nucleic acid molecule having the nucleotide sequence shownin FIG. 1 [SEQ ID NO:1] or the nucleotide sequence of the IL-19 cDNAcontained in the above-described deposited clone, or nucleic acidmolecule having a sequence complementary to one of the above sequences.Such isolated molecules, particularly DNA molecules, are useful asprobes for gene mapping by in situ hybridization with chromosomes andfor detecting expression of the IL-19 gene in human tissue, forinstance, by Northern blot analysis.

[0034] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in SEQ ID NO:1 isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700,725, 750, 775, 800, 825, 850, 875, 900, 925 or 950 nt in length are alsouseful according to the present invention as are fragments correspondingto most, if not all, of the nucleotide sequence of the deposited cDNA oras shown in SEQ ID NO:1. By a fragment at least 20 nt in length, forexample, is intended fragments which include 20 or more contiguous basesfrom the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in SEQ ID NO:1.

[0035] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC Deposit No. 97662. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0036] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0037] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1). Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3′ terminal poly(A) tract of the IL-19 cDNA shown in SEQ ID NO:1), or toa complementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

[0038] Since an IL-19 cDNA clone has been deposited and its determinednucleotide sequence is provided in FIG. 1 [SEQ ID NO:1], generatingpolynucleotides which hybridize to a portion of the IL-19 cDNA moleculewould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication of the IL-19 cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the IL-19 cDNA molecule.Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the IL-19 cDNA shown in FIG. 1[SEQ ID NO:1]), or to a complementary stretch of T (or U) resides, wouldnot be included in a polynucleotide of the invention used to hybridizeto a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule contain apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0039] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the IL-19protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about −5 to about 4 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 64 to about 82in SEQ ID NO:2; and a polypeptide comprising amino acid residues fromabout 115 to about 125 in SEQ ID NO:2. The inventors have determinedthat the above polypeptide fragments are antigenic regions of the IL-19protein. Methods for determining other such epitope-bearing portions ofthe IL-19 protein are described in detail below.

[0040] As indicated, nucleic acid molecules of the present inventionwhich encode the IL-19 polypeptide may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 24 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing—including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.Proc. Natl. Acad. Sci., USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767-768(1984). As discussed below, other such fusion proteins include the IL-19fused to Fc at the N- or C-terminus.

[0041] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the IL-19 protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, ed. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

[0042] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the IL-19 protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions.

[0043] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 90% identical, and more preferably at least 95%, 96%, 97%, 98%or 99% identical to a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 153 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97662; (e) a nucleotidesequence encoding the mature IL-19 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97662;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d) or (e).

[0044] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding anIL-19 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the IL-19polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0045] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIG. 1 or to the nucleotidessequence of the deposited cDNA clone can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711.Bestfit uses the local homology algorithm of Smith and Waterman(Advances in Applied Mathematics 2: 482-489, 1981) to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0046] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 [SEQ ID NO:1] or to the nucleic acid sequenceof the deposited cDNA, irrespective of whether they encode a polypeptidehaving IL-19 activity. This is because, even where a particular nucleicacid molecule does not encode a polypeptide having IL-19 activity, oneof skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having IL-19 activityinclude, inter alia, (1) isolating the IL-19 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe IL-19 gene as described in Verma et al., Human Chromosomes: a Manualof Basic Techniques, Pergamon Press, New York (1988); and (3) NorthernBlot analysis for detecting IL-19 mRNA expression in specific cell types(e.g., activated monocytes).

[0047] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 [SEQ ID NO:1] or to the nucleic acid sequenceof the deposited cDNA which do, in fact, encode a polypeptide havingIL-19 protein activity. By “a polypeptide having IL-19 activity” isintended polypeptides exhibiting activity similar, but not necessarilyidentical, to an activity of the IL-19 protein of the invention (eitherthe full-length protein or, preferably, the mature protein) as measuredin a particular biological assay.

[0048] IL-19 exhibits several biological activities which could form thebasis of such biological assays. In particular, it is believed by theinventors that IL-19 has the property of modulating the synthesis of atleast one cytokine in the group consisting of IFN-γ, lymphotoxin, IL-2,IL-3, and GM-CSF in a population of T helper cells induced to synthesizeone or more of these cytokines by exposure to syngeneicantigen-presenting cells (APCs) and antigen. In this activity, APCs aretreated so that they become incapable of replication, but theirantigen-processing machinery remains functional. This is convenientlyaccomplished by irradiating the APCs, e.g. with about 1500-3000 R (gammaor X-radiation) before mixing with the T-cells.

[0049] Alternatively, changes in levels of cytokine production may beassayed in primary or, preferably, secondary mixed-lymphocyte reactions(MLR), in which case syngeneic APCs need not be used. MLRs arewell-known in the art, e.g., Bradley, pp 162-166 in Mishell et al., eds.Selected Methods in Cellular Immunology (Freeman, San Francisco, 1980);and Battisto, et al., Meth, in Enzymol. 150:83-91 (1987). Briefly, thecell populations are mixed, one of the populations having been treatedprior to mixing to prevent proliferation, e.g., by irradiation.Preferably, the cell populations are prepared at a concentration ofabout 2×10⁶ cells/ml in supplemented media, e.g. RPMI 1640 with 10%fetal calf serum. For both controls and test cultures, mix 0.05 ml ofeach population for the assay. For a secondary MLR, the cells remainingafter 7 days in the primary MLR are re-stimulated by freshly prepared,irradiated stimulator cells. The sample suspected of containing IL-19may be added to the test cultures at the time of mixing, and bothcontrols and test cultures may be assayed for cytokine production from1-3 days after mixing.

[0050] Obtaining T cell populations and/or APC populations for IL-19assays employs techniques well known in the art which are fullydescribed in DiSabato et a;., eds., Meth. in Enzymol. Vol. 108 (1984).APCs for the preferred IL-19 assay are peripheral blood monocytes. Theseare obtained using standard techniques, e.g. as described by thefollowing articles in the aforementioned DiSabato et a;., eds., Meth. inEnzymol. Vol. 108 (1984): Boyum, pp. 88-102; Mage, pp. 118-132; Litvinet al., pp. 298-302; Stevenson, pp. 242-249; and Romain, pp. 148-153,which references are herein incorporated by reference. Preferably,helper T-cells are used in the IL-19 assays, which are obtained by firstseparating lymphocytes from the peripheral blood, and then selecting,e.g., by panning or flow cytometry, helper cells using a commerciallyavailable anti-CD4 antibody, e.g. OKT4, described in U.S. Pat. No.4,381,295, and available form Ortho Pharmaceutical Corp. The requisitetechniques are fully disclosed by Boyum in Scand. JL. Clin. Lab.Invest., 21(Suppl. 97):77 (1968) and in Meth. in Enzymol. Vol. 108(1984) (cited above) and by Bram et al., Meth. in Enzymol. 121:737-748(1986). Generally, PBLs are obtained from fresh blood by Ficoll-Hypaquedensity gradient centrifugation.

[0051] A variety of antigens can be employed in the assay, e.g. Keyholelimpet hemocyanin (KLH), fowl γ-globulin, or the like. More preferably,in place of antigen, helper T cells are stimulated with anti-CD3monoclonal antibody, e.g. OKT3 disclosed in U.S. Pat. No. 4,361,549, inthe assay.

[0052] Cytokine concentrations in control and test samples are measuredby standard biological and/or immunochemical assays. Construction ofimmunochemical assays for specific cytokines is well known in the artwhen the purified cytokine is available; e.g. Campbell, MonoclonalAntibody Technology (Elsevier, Amsterdam, 1984); Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and U.S. Pat.No. 4,486,530, are examples of the extensive literature on the subject.ELISA kits for human IL-2, human IL-3, and human GM-CSF are commerciallyavailable from Genzyme Corp. (Boston, Mass.); and an ELISA kit for humanIFN-γ is commercially available from Endogen, Inc. (Boston, Mass.).Polyclonal antibodies specific for human lymphotoxin are available fromgenzyme, Corp., which can be used in a radioimmunoassay for humanlymphotoxin, e.g., Chard, An Introduction to Radioimmunoassay andRelated Techniques (Elsevier, Amsterdam, 1982).

[0053] Biological assays of the cytokines listed above can also be usedto determine whether a sample has IL-19 activity, i.e, whether a samplemodulates cytokine expression or activity in a manner similar to IL-19.A biological assay for human lymphotoxins disclosed by Aggarwal, Meth.in Enzymol. 116:441-447 (1985), and by Matthews et al., in Lymphokinesand Interferons: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C., 1987, pp. 221-225. Human IL-2 and GM-CSF can beassayed with factor-dependent cell lines CTLL-2 and KG-1, available fromthe ATCC under accession numbers TIB 214 and CCL246, respectively. HumanIL-3 can be assayed by its ability to simulate the formation of a widerange of hematopoietic cell colonies in soft agar cultures, e.g. asdescribed by MEtcalf, The Hematopoietic Colony Stimulating Factors(Elsevier, Amsterdam, 1984). IFN-γ can be quantified with anti-viralassays, e.g. Meager, pp. 129-147, in Clemens et al., eds (cited above).

[0054] Cytokine production can also be determined by mRNA analysis.Cytokine mRNAs can be measured by cytoplasmic dot hybridization, asdescribed by White et al. (J. Biol. Chem. 257:8569-8572 (1982)) andGillespie et al., U.S. Pat. No. 4,483,920, both of which are herebyincorporated by reference. Other approaches include dot blotting usingpurified RNA, e.g. Chapter 6 in Hames et al., eds, Nucleic AcidHybridization: A Practical Approach, IRL PRess, Washington D.C., 1985.

[0055] Some samples to be tested for IL-19 activity must be pretreatedto remove cytokines that might interfere with the assay. For example,IL-2 increases the production of IFN-γ in some cells. Thus, depending onthe T helper cells used in the assay, IL-2 may have to be removed fromthe sample being tested. Such removals are conveniently accommodated bypassing a sample over a standard anti-cytokine affinity column.

[0056] Thus, by using an assay such as those described above, the effectof the substance suspected of having IL-19 activity on the activity ofany one of a number of cytokines may be compared to the IL-19 protein ofthe invention, in order to determine if the sample indeed has IL-19activity.

[0057] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in FIG. 1 [SEQ ID NO: 1] willencode a polypeptide “having IL-19 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming one of the above-described comparison assays. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having IL-19 protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).

[0058] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U., et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that were surprisingly tolerant of amino acid substitutions.

Vectors and Host Cells

[0059] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof IL-19 polypeptides or portions thereof by recombinant techniques.

[0060] Recombinant constructs may be introduced into host cells usingwell known techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0061] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0062] Preferred are vectors comprising cis-acting control regions tothe polynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

[0063] In certain preferred embodiments in this regard, the vectorsprovide for specific expression, which may be inducible and/or celltype-specific. Particularly preferred among such vectors are thoseinducible by environmental factors that are easy to manipulate, such astemperature and nutrient additives.

[0064] Expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophage, yeast episomes, yeastchromosomal elements, viruses such as baculoviruses, papova viruses,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

[0065] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0066] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts includebacterial cells, such as E. coli, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells; insect cells suchas Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COSand Bowes melanoma cells; and plant cells. Appropriate culture media andconditions for the above-described host cells are known in the art.

[0067] Among vectors preferred for use in bacteria include pA2, pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

[0068] Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

[0069] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986).

[0070] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0071] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0072] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fe part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFe part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Feportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition 8:52-58 (1995) andK. Johanson et al., The Journal of Biological Chemistry270(16):9459-9471 (1995).

[0073] The IL-19 protein can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

[0074] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

IL-19 Polypeptides and Peptides

[0075] The invention further provides an isolated IL-19 polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in FIG. 1 [SEQ ID NO:2], or a peptide or polypeptidecomprising a portion of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of at least to amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

[0076] It will be recognized in the art that some amino acid sequence ofthe IL-19 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

[0077] Thus, the invention further includes variations of the IL-19polypeptide which show substantial IL-19 polypeptide activity or whichinclude regions of IL-19 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions. As indicated above, guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin Bowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

[0078] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

[0079] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the IL-19 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

[0080] The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-γ to only one of the two known types of TNF receptors. Thus, theIL-19 of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

[0081] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0082] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of substitutions for any givenIL-19 polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or3.

[0083] Amino acids in the IL-19 protein of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

[0084] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the IL-19 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0085] The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein); a polypeptide comprising amino acids about −24 toabout 153 in SEQ ID NO:2; a polypeptide comprising amino acids about −23to about 153 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 153 in SEQ ID NO:2; as well as polypeptides which are atleast 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98% or 99% identical to thosedescribed above and also include portions of such polypeptides with atleast 30 amino acids and more preferably at least 50 amino acids.

[0086] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of an IL-19polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the IL-19 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0087] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1 [SEQ ID NO:2] or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0088] The polypeptide of the present invention is useful as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

[0089] As described in detail below, the polypeptides of the presentinvention can be used to raise polyclonal and monoclonal antibodies,which are useful in diagnostic assays for detecting IL-19 proteinexpression as described below or as agonists and antagonists capable ofenhancing or inhibiting IL-19 protein function. Further, suchpolypeptides can be used in the yeast two-hybrid system to “capture”IL-19 protein binding proteins which are also candidate agonist andantagonist according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340:245-246 (1989).

[0090] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. These immunogenicepitopes are believed to be confined to a few loci on the molecule. Onthe other hand, a region of a protein molecule to which an antibody canbind is defined as an “antigenic epitope.” The number of immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, for instance, Geysen, H. M., Meloen, R. H. and Barteling,S. J. (1984) Use of peptide synthesis to probe viral antigens forepitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci.USA 81:3998-4002 (1983).

[0091] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals.

[0092] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.See, for instance, Wilson, et al., Cell 37:767-778 at 777.

[0093] Antigenic epitope-bearing peptides and polypeptides of theinvention designed according to the above guidelines preferably containa sequence of at least seven, more preferably at least nine and mostpreferably between about 15 to about 30 amino acids contained within theamino acid sequence of a polypeptide of the invention.

[0094] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate IL-19-specific antibodies include: a polypeptidecomprising amino acid residues from about −5 to about 4 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 64 to about 82in SEQ ID NO:2; and a polypeptide comprising amino acid residues fromabout 115 to about 125 in SEQ ID NO:2. As indicated above, the inventorshave determined that the above polypeptide fragments are antigenicregions of the IL-19 protein.

[0095] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. Houghten, R. A. (1985)General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0096] As one of skill in the art will appreciate, IL-19 polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric IL-19 protein or proteinfragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

[0097] The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference

Chromosome Assays

[0098] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0099] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an interleukin-19 proteingene. This can be accomplished using a variety of well known techniquesand libraries, which generally are available commercially. The genomicDNA then is used for in situ chromosome mapping using well knowntechniques for this purpose. Typically, in accordance with routineprocedures for chromosome mapping, some trial and error may be necessaryto identify a genomic probe that gives a good in situ hybridizationsignal.

[0100] In some cases, in addition, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedportion.

[0101] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of portions from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0102] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES,Pergamon Press, New York (1988).

[0103] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, MENDELIAN INHERITANCE IN MAN, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0104] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0105] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0106] Treatment of Pathological Conditions by IL-19 Inhibition ofCytokine Production

[0107] As noted above, IL-19 is secreted by activated monocytes, andshares significant homology with human IL-10. Thus, it is believed bythe inventors that IL-19 is active in inhibiting cytokine productionduring the mammalian immune response. The cytokines whose production maybe affected by IL-19 include IFN-γ, TNF-α, and IL-6.

[0108] One such activity of IL-19 is the ability to limit excessiveproduction of gamma-interferon (IFN-γ), and hence the consequent effectsof such production, including major histocompatibility (MHC) associatedauto-immune diseases. As increased IFN-γ expression has been implicatedin an increase of MHC genes, which may then increase the chance of anautoimmune response against the MHC-overexpressing cells, the ability tolimit IFN-γ expression may be therapeutically valuable in the treatmentof clinical manifestations of such MHC disorders. These includerheumatoid arthritis, systemic lupus erythematosus (SLE), myastheniagravis, insulin-dependent diabetes mellilitus, and thyroiditis.

[0109] The down regulation of IFN-γ by IL-19 may also be therapeuticallyvaluable in treating parasitic infections such as leishmaniasis. Levelsof IFN-γ, IL-2 and IL-4 are all involved in the regulation of the lifecycle of this parasite. Thus, the ability to regulate the production ofthese cytokines by IL-19 will be therapeutically valuable.

[0110] Given the activities modulated by IL-19, it is readily apparentthat a substantially altered (increased or decreased) level ofexpression of IL-19 in an individual compared to the standard or“normal” level produces pathological conditions such as those describedabove. It will also be appreciated by one of ordinary skill that, sincethe IL-19 protein of the invention is translated with a leader peptidesuitable for secretion of the mature protein from the cells whichexpress IL-19, when IL-19 protein (particularly the mature form) isadded from an exogenous source to cells, tissues or the body of anindividual, the protein will exert its modulating activities on any ofits target cells. Therefore, it will be appreciated that conditionscaused by a decrease in the standard or normal level of IL-19 activityin an individual, can be treated be administration of IL-19 protein.Thus, the invention further provides a method of treating an individualin need of an increased level of IL-19 activity comprising administeringto such an individual a pharmaceutical composition comprising an amountof an isolated IL-19 polypeptide of the invention, particularly a matureform of the IL-19 protein of the invention, effective to increase theIL-19 activity level in such an individual.

[0111] One of ordinary skill will appreciate that effective amounts ofthe IL-19 polypeptides for treating an individual in need of anincreased level of IL-19 activity can be determined empirically for eachcondition where administration of IL-19 is indicated. The polypeptidehaving IL-19 activity my be administered in pharmaceutical compositionsin combination with one or more pharmaceutically acceptable excipients.It will be understood that, when administered to a human patient, thetotal daily usage of the pharmaceutical compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the type and degree of the response to be achieved; thespecific composition an other agent, if any, employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thecomposition; the duration of the treatment; drugs (such as achemotherapeutic agent) used in combination or coincidental with thespecific composition; and like factors well known in the medical arts.

[0112] For example, it is predicted that satisfactory results areobtained by oral administration of a polypeptide having IL-19 activityin dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or,in divided doses, 2 to 4 times per day. On administration parenterally,for example by i.v. drip or infusion, dosages on the order of from 0.01to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1to 1.0 mg/kg/day can be used. Suitable daily dosages for patients arethus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mgp.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50mg i.v.

[0113] Dosaging may also be arranged in a patient specific manner toprovide a predetermined concentration of an IL-19 activity in the blood,as determined by an RIA technique, for instance. Thus patient dosagingmay be adjusted to achieve regular on-going trough blood levels, asmeasured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150to 500 ng/ml.

[0114] Pharmaceutical compositions of the invention may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0115] Pharmaceutical compositions of the present invention forparenteral injection can comprise pharmaceutically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsionsas well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use. Examples of suitable aqueousand nonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

[0116] The compositions of the present invention may also containadjuvants such as preservatives, wetting agents, emulsifying agents, anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents such as sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

[0117] In some cases, in order to prolong the effect of thepharmaceutical composition, it is desirable to slow the absorption ofthe drug from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe drug then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered drug form is accomplished bydissolving or suspending the drug in an oil vehicle.

[0118] Injectable depot forms are made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

[0119] The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

[0120] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compounds are mixed with at least one item pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f)-absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate,h)-absorbents such as kaolin and bentonite clay, and i) lubricants suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and mixtures thereof. In the case ofcapsules, tablets and pills, the dosage form may also comprise bufferingagents.

[0121] Solid compositions of a similar type may also be employed asfillers in soft and hard filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0122] The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

[0123] The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

[0124] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isoptopyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

[0125] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0126] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

[0127] The active polypeptide can also be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to theagent or inhibitor, stabilizers, preservatives, excipients, and thelike. The preferred lipids are the phospholipids and the phosphatidylcholates (lecithins), both natural and synthetic. Methods to formliposomes are known in the art. See, for example, Prescott, Ed., Methodsin Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33 et seq.

[0128] Use of IL-19 in Adoptive Immunotherapy of Cancer

[0129] Another therapeutic application of IL-19 is the administration ofIL-19 in adoptive immunotherapy to prevent or reduce the production ofcytokines believed to be responsible for many of the deleterious sideeffects currently encountered in adoptive immunotherapy. As used herein,“Adoptive immunotherapy” means therapy wherein functionalcancer-fighting immune cells are transferred to a patient. Thecancer-fighting immune cells preferably comprise tumor infiltratinglymphocytes (TILs) originating from the patient himself. As is known inthe art, while IL-2 is useful in adoptive immunotherapy due to itsability to activate the killer cells transferred to the patent(Rosenberg et al., Ann. Rev. Immunol. 4: 681-709 (1988)), the severeside effects caused directly or indirectly by IL-2 have been an obstacleto the development of routine treatment protocols based on this approach(Hsu, D-H et al., WO 92/12726). As IL-19 is believed to be capable ofpreventing or reducing the production of cytokines responsible for theseside effects, TILs cultured in the presence of both IL-2 and IL-19 priorto administration, wherein the administration of IL-2 and IL-19 iscontinued after the administration of those TILs to the patient, mayreduce the deleterious side effects typical of adoptive immunotherapy.

[0130] Use of IL-19 Antagonists in the Restoration of Immunocompetencyto T Helper Cells in HIV-Infected Patients

[0131] Another therapeutic application of the IL-19 polypeptide of theinvention is the use of the polypeptide to identify IL-19 antagonists,such as an antibody specific for binding to IL-19, which can then beused to increase the production of IL-2 in T helper cells. For example,patients infected with human immunodeficiency virus (HIV) have adecreased level of IL-2 production in non-virally infected T helpercells. IL-19 is believed to be capable of preventing or reducing theproduction of IL-2. Therefore, 4he administration of IL-19 antagonistsmay result in an increase in IL-2 production in HIV-infected patients.As IL-2 is responsible for T cell proliferation, the maintenance of IL-2production is beneficial to HIV-infected patients.

[0132] Antagonists specific for IL-19 can be made by mutating the aminoacid sequence of IL-19 using standard mutagenesis methods well known tothose of ordinary skill in the art. Such methods include the use of M13vectors to introduce single-site mutations, to delete random amino acidsfrom IL-19, or to add amino acids. The resulting muteins are then testedin standard assays for the ability to compete with non-mutated IL-19,including assays which test the ability of the mutein, as compared withthe IL-19 protein of the invention, to enhance IL-2 dependentproliferation of T cells in vitro.

[0133] Other suitable IL-19 antagonists include an antibody specific forbinding to IL-19 (αIL-19) which interferes with its binding to the Thelper receptor. Production of such antibodies has been described infull above.

[0134] The antibodies used in the method of the invention preferably areautologous for the patient, thereby minimizing further immunologicalproblems. However, as immunodeficient individuals in need of thistreatment will tend to be less reactive to non-self antibodies, non-selfantibodies derived from cells of the same species will also be useful.

[0135] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of IL-19 in E. coli

[0136] The DNA sequence encoding the mature IL-19 protein in thedeposited cDNA clone was amplified using PCR oligonucleotide primersspecific to the amino terminal sequences of the IL-19 protein and tovector sequences 3′ to the gene. Additional nucleotides containingrestriction sites to facilitate cloning were added to the 5′ and 3′sequences respectively.

[0137] The 5′ oligonucleotide primer had the sequence 5′ GGC ATG CCA TGGAGT TAC AGT GTG TIT CCC 3′ [SEQ ID NO:4] (sequences specific to theIL-19 nucleotide sequence are underlined), and included an NcoIrestriction site.

[0138] The 3′ primer had the sequence 5′ GGA AGA TCT AGC TGA GGA CAT TAC3′ [SEQ ID NO:5] containing the underlined 15 nucleotides complementaryto the last 15 nucleotides immediately after the IL-19 protein codingsequence in FIG. 1. The 3′ primer included a BglII restriction site.

[0139] The restriction sites were convenient to restriction enzyme sitesin the bacterial expression vector pQE60, which were used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites. The amplified IL-19 protein DNAand the vector pQE60 both were digested with NcoI and BglII and thedigested DNAs were then ligated together. Insertion of the IL-19 proteinDNA into the restricted pQE60 vector placed the IL-19 protein codingregion downstream of and operably linked to the vector's IPTG-induciblepromoter and in-frame with an initiating ATG appropriately positionedfor translation of IL-19 protein.

[0140] The ligation mixture was transformed into competent E. coil cellsusing standard procedures. Such procedures are described in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”),was used in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing IL-19protein, is available commercially from Qiagen.

[0141] Transformants were identified by their ability to grow on LBplates in the presence of ampicillin and kanarnycin. Plasmid DNA wasisolated from resistant colonies and the identity of the cloned DNA wasconfirmed by restriction analysis.

[0142] Clones containing the desired constructs were grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 g/ml).

[0143] The O/N culture was used to inoculate a large culture, at adilution of approximately 1:100 to 1:250. The cells were grown to anoptical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 0.1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently were incubated further for 4 hours (FIG. 2 shows IL-19protein induction; samples removed 0, 3, 5, 6, and 24 hours after theaddition of IPTG were run on a 12.5% polyacrylamide gel and stained withbrilliant blue). The mobility of the IL-19 protein is indicated by anarrow. Cells were then harvested by centrifugation and disrupted bygentle shaking overnight in 6M guanidine HCl in 50 mM NaPO₄ buffer at pH8.0 at 4° C. The lysate was then centrifuged and passed over a SepharoseCL-4B (Pharmacia) column. The flowthrough was then passed over a columncontaining activated Ni²⁺-NTA-agarose (Qiagen). IL-19 protein wascollected from the column in a fraction consisting of 6M guanidine HClpH 5.0. Guanidine HCl was removed from the IL-19-containing fraction bydialysis against successively reduced concentrations of guanidine inphosphate-buffered saline (PBS) at pH 5.5.

Example 2 Cloning and Expression of IL-19 in a Baculovirus ExpressionSystem

[0144] The cDNA sequence encoding the full length IL-19 protein in thedeposited clone was amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

[0145] The 5′ primer had the sequence 5′ GGC GGG ATC CCG CCA TCA TGA AGTTAC AGT GTG TTT CCC 3′ [SEQ ID NO:6] containing the underlined BamHIrestriction enzyme site followed by 29 bases of the sequence of IL-19protein in FIG. 1. Inserted into an expression vector, as describedbelow, the 5′ end of the amplified fragment encoding IL-19 provided anefficient signal peptide. An efficient signal for initiation oftranslation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196: 947-950 (1987) is appropriately located in the vector portionof the construct.

[0146] The 3′ primer had the sequence 5′CCC AAG CTT GGT ACC TCA TCA AGCTGA GGA CAT TAC 3′ [SEQ ID NO:7] containing the underlined Asp718restriction site followed by nucleotides complementary to the last 21nucleotides of the IL-19 coding sequence set out in FIG. 1.

[0147] The amplified fragment was isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then was digested with BamHI and Asp718 and againwas purified on a 1% agarose gel. This fragment is designated herein F2.

[0148] The vector and pA2-GP were used to express the IL-19 protein inthe baculovirus expression system, using standard methods, as describedin Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECTCELL CULTURE PROCEDURES, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites. The signalpeptide of AcMNPV gp67, including the N-terminal methionine, is locatedjust upstream of a BamHI site. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For an easyselection of recombinant virus the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedrin promoter and isfollowed by the polyadenylation signal of the polyhedrin gene. Thepolyhedrin sequences are flanked at both sides by viral sequences forcell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide. The IL-19protein was also expressed in baculovirus using the pA2 vector.

[0149] Many other baculovirus vectors could be used in place of pA2-GP,such as pAc373, pVL941 and pAcIM1 provided, as those of skill readilywill appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

[0150] The plasmid was digested with the restriction enzymes BamHI andAsp718 and then was dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA was then isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean”BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein“V2”.

[0151] Fragment F2 and the dephosphorylated plasmid V2 were ligatedtogether with T4 DNA ligase. E. coli HB101 cells were transformed withligation mix and spread on culture plates. Bacteria were identified thatcontained the plasmid with the human IL-19 gene by digesting DNA fromindividual colonies using BamHI and Asp718 and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment was confirmed by DNA sequencing. This plasmid is designatedherein pBacIL-19.

[0152] 5 μg of the plasmid pBacIL-19 was co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBacCKP-15 were mixed in a sterile well of a microtiter plate containing50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate was rocked back and forthto mix the newly added solution. The plate was then incubated for 5hours at 27° C. After 5 hours the transfection solution was removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum was added. The plate was put back into an incubator andcultivation is continued at 27° C. for four days.

[0153] After four days the supernatant was collected and a plaque assayis performed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) was used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

[0154] Four days after serial dilution, the virus was added to thecells. After appropriate incubation, blue stained plaques were pickedwith the tip of an Eppendorf pipette. The agar containing therecombinant viruses was then resuspended in an Eppendorf tube containing200 μl of Grace's medium. The agar was removed by a brief centrifugationand the supernatant containing the recombinant baculovirus was used toinfect Sf9 cells seeded in 35 mm dishes. Four days later thesupernatants of these culture dishes were harvested and then they werestored at 4° C. A clone containing properly inserted hESSB I, II and IIIwere identified by DNA analysis including restriction mapping andsequencing. This is designated herein as V-IL-19.

[0155] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-IL-19 at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium was removed and wasreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of35S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) wereadded. The cells were further incubated for 16 hours and then they wereharvested by centrifugation, lysed and the labeled proteins werevisualized by SDS-PAGE and autoradiography.

Example 3 In Vitro Transcription/Translation of IL-19 Protein

[0156] Recombinant IL-19 proteins were prepared using the TNT CoupledWheat Germ Extract System (Promega, Madison, Wis.).

[0157] 25 μl of TNT wheat germ extract, 10 U T3 RNA polymerase, 1 mMamino acid mixture (methionine-free), 4 μCi ³⁵S-methionine (1000Ci/mmol), 40 U RNasin (Promega, Madison, Wis.), and 1 μg template DNAwere combined in a final volume of 50 μl and incubated at 30° C. for 2h. Coupled transcription/translation reactions included the followingtemplate DNAs: (1) No DNA, (2) pBluescript, (3) nucleotides 44-577(corresponding to amino acids 1-177) of the IL-19 sequence shown in FIG.1 cloned into pBluescript, (4) nucleotides 116-577 (corresponding toamino acids 25-177) OF THE IL-19 coding sequence cloned intopBluescript, (5) a gel-purified PCR product derived from the templatedescribed in (3) and amplified using M13 forward and reverse primers ina standard PCR reaction. Samples were heated to 95° C. for 10 minutesand a 5 μl aliquot of each sample was then loaded on to a 15%polyacrylamide gel. The gel was run at 100 volts for approximately 2hours. The gel was then dried and exposed to X-ray film for 3 days atroom temperature. The mobility of full-length and truncated (no signalsequence) IL-19 proteins are indicated in FIG. 3. An apparent molecularmass marker (M) shows the relative mobilities of 14.3, 21.5, 30, 46, 66,97.4, and 220 kD proteins.

Example 4 Cloning and Expression in Mammalian Cells

[0158] Most of the vectors used for the transient expression of theIL-19 protein gene sequence in mammalian cells should carry the SV40origin of replication. This allows the replication of the vector to highcopy numbers in cells (e.g. COS cells) which express the T antigenrequired for the initiation of viral DNA synthesis. Any other mammaliancell line can also be utilized for this purpose.

[0159] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftrancription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular signals can also be used (e.g. human actin promoter).Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109). Mammalian host cells that could be used include, human Hela,283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 andCV1, African green monkey cells, quail QC1-3 cells, mouse L cells andChinese hamster ovary cells.

[0160] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

[0161] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase) is auseful marker to develop cell lines that carry several hundred or evenseveral thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) cells are often used for theproduction of proteins.

[0162] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-4470 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 4(a) Cloning and Expression in COS Cells

[0163] The expression plasmid, pIL-19 HA, is made by cloning a cDNAencoding IL-19 into the expression vector pcDNA4 (which can be obtainedfrom Invitrogen, Inc.).

[0164] The expression vector pcDNA4 contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

[0165] A DNA fragment encoding the IL-19 protein and an HA tag fused inframe to its 3′ end is cloned into the polylinker region of the vectorso that recombinant protein expression is directed by the CMV promoter.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein described by Wilson et al., Cell 37: 767 (1984).The fusion of the HA tag to the target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

[0166] The plasmid construction strategy is as follows. The IL-19 cDNAof the deposited clone is amplified using primers that containconvenient restriction sites, much as described above regarding theconstruction of expression vectors for expression of IL-19 in E. coli.To facilitate detection, purification and characterization of theexpressed IL-19, one of the primers contains a hemagglutinin tag (“HAtag”) as described above.

[0167] Suitable primers include the following, which are used in thisexample. The 5′ primer, containing the underlined BamHI site, an AUGstart codon and 22 bp of the 5′ coding region has the followingsequence: (SEQ ID NO:8) 5′GGC GGG ATC CCG CCA TGA AGT TAC AGT GTG TTTCCC 3′.

[0168] The 3′ primer, containing the underlined Asp 718 site, a stopcodon, 10 codons thereafter forming the hemagglutinin HA tag, and 25 bpof 3′ coding sequence (at the 3′ end) has the following sequence: (SEQID NO:9) 5′CCC AAG CTT GGT ACC TCA TCA GAA AGC GTA GTC TGG GAC GTC GTATGG GTA AGC TGA GGA CAT TAC TTC ATG ATT C 3′.

[0169] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with HindIII and XhoI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the IL-19-encoding fragment.

[0170] For expression of recombinant IL-19, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression ofIL-19 by the vector.

[0171] Expression of the IL-19/HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4(b) Cloning and Expression in CHO Cells

[0172] The vector pC4 is used for the expression of IL-19 protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.37146]. Both plasmids contain the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

[0173] Plasmid pC4 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-4470) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530, 1985). Downstream of the promoter are the followingsingle restriction enzyme cleavage sites that allow the integration ofthe genes: BamHI, Pvull, and Nrul. Behind these cloning sites theplasmid contains translational stop codons in all three reading framesfollowed by the 3′ intron and the polyadenylation site of the ratpreproinsulin gene. Other high efficient promoters can also be used forthe expression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

[0174] Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0175] The plasmid pC4 is digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0176] The DNA sequence encoding IL-19 protein is amplified using PCRoligonucleotide primers specific to the amino terminal sequence of theIL-19 protein and to carboxy terminal sequence 3′ to the gene.Additional nucleotides containing restriction sites to facilitatecloning are added to the 5′ and 3′ sequences respectively.

[0177] The 5′ primer has the sequence 5′ GGC GGG ATC CCG CCA TCA TGA AGTTAC AGT GTG TTT CCC 3′ (SEQ ID NO: 10), containing the underlined BamHIrestriction enzyme site followed by Kozak sequence and 23 bases of thesequence of IL-19 in FIG. 1. The 3′ primer has the sequence 5′CCC AAGCTT GGT ACC TCA TCA AGC TGA GGA CAT TAC 3′ (SEQ ID NO: 11), containingthe underlined Asp 718 restriction site followed by nucleotidescomplementary to 15 bp of the nucleotide sequence preceding the stopcodon in FIG. 1. The restrictions sites are convenient to restrictionenzyme sites in the CHO expression vector pC4.

[0178] The amplified fragments are isolated from a 1% agarose gel asdescribed above and then digested with the endonucleases BamHI andAsp718 and then purified again on a 1% agarose gel.

[0179] The isolated fragment and the dephosphorylated vector are thenligated with T4 DNA ligase. E. coli HB101 cells are then transformed andbacteria identified that contained the plasmid pC4 inserted in thecorrect orientation using the restriction enzyme BamHI. The sequence ofthe inserted gene is confirmed by DNA sequencing.

[0180] Transfection of CHO-DHFR-Cells

[0181] Chinese hamster ovary cells lacking an active DHFR enzyme areused for transfection. 5 μg of the expression plasmid pC4 arecotransfected with 0.5 μg of the plasmid pSVneo using the lipofectingmethod (Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated from 10-14 days. After this period,single clones are trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25 nM, 50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 μM, 21 μM, 5 μM, 10 mM, 20 mM).The same procedure is repeated until clones grow at a concentration of100 μM-200 μM.

[0182] The expression of the desired gene product is analyzed by Westernblot analysis and SDS-PAGE or by reverse phase HPLC analysis.

Example 5 Tissue Distribution of IL-19 Protein Expression

[0183] Northern blot analysis was carried out to examine the levels ofexpression of IL-19 protein in human tissues, using methods describedby, among others, Sambrook et al, cited above.

[0184] HL-60, THP-1 and U937 cell lines and primary human monocytesisolated by adherence from a mixed leukocyte population were grown for12 hours either in the presence (+) or absence (−) of bacteriallipopolysaccharide (LPS). Total RNA was prepared from the cultures withTRIzol Reagent (Life Technologies, Gaithersburg, Md.) essentially asdescribed by the manufacturer. Total RNA (10 μg) was dried completely,resuspended in a formamide/formaldehyde loading buffer, and resolved byelectrophoresis through a 1% agarose gel containing 2.2 M formaldehyde.The gel was transferred overnight in 20×SSC to a nylon membrane(Boehringer Mannheim, Indianapolis, Ind.). Probe DNA was prepared by PCRamplifying the entire IL-19 insert shown in FIG. 1 using M13 Forward andReverse primers. Probe DNA (25 ng) was labeled with 32P using theRediPrime Random Primer Labeling Kit (Amersham Life Science) to aspecific activity of greater than 10⁶ CPM/ng. The blot was hybridizedwith denatured probe in a10 ml Hybrizol hybridization solution overnightat 42° C. The blot was then washed in approximately 100 ml of0.2−SSC/0.1% SDS at 25° C. for 20 minutes, and then twice inapproximately 100 ml of 0.2×SSC/0.1% SDS at 65° C. for 15 min. The blotwas then exposed to x-ray film for 5 days, and is shown in FIG. 4. Thearrow shows the migration of the IL-19-specific RNA. An RNA marker showsthe relative mobilities of 0.24, 1.35, 2.37, 4.40, 7.46, and 9.49 kbRNAs.

[0185] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0186] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0187] The disclosures of all patents, patent applications, andpublications referred to herein are hereby incorporated by reference.

1 17 1 966 DNA Homo sapiens CDS (44)..(574) sig_peptide (44)..(115)mat_peptide (116)..(574) 1 ggcacgagca caaggagcag cccgcaagca ccaagtgagaggc atg aag tta cag 55 Met Lys Leu Gln tgt gtt tcc ctt tgg ctc ctg ggtaca ata ctg ata ttg tgc tca gta 103 Cys Val Ser Leu Trp Leu Leu Gly ThrIle Leu Ile Leu Cys Ser Val -20 -15 -10 -5 gac aac cac ggt ctc agg agatgt ctg att tcc aca gac atg cac cat 151 Asp Asn His Gly Leu Arg Arg CysLeu Ile Ser Thr Asp Met His His -1 1 5 10 ata gaa gag agt ttc caa gaaatc aaa aga gcc atc caa gct aag gac 199 Ile Glu Glu Ser Phe Gln Glu IleLys Arg Ala Ile Gln Ala Lys Asp 15 20 25 acc ttc cca aat gtc act atc ctgtcc aca ttg gag act ctg cag atc 247 Thr Phe Pro Asn Val Thr Ile Leu SerThr Leu Glu Thr Leu Gln Ile 30 35 40 att aag ccc tta gat gtg tgc tgc gtgacc aag aac ctc ctg gcg ttc 295 Ile Lys Pro Leu Asp Val Cys Cys Val ThrLys Asn Leu Leu Ala Phe 45 50 55 60 tac gtg gac agg gtg ttc aag gat catcag gag cca aac ccc aaa atc 343 Tyr Val Asp Arg Val Phe Lys Asp His GlnGlu Pro Asn Pro Lys Ile 65 70 75 ttg aga aaa atc agc agc att gcc aac tctttc ctc tac atg cag aaa 391 Leu Arg Lys Ile Ser Ser Ile Ala Asn Ser PheLeu Tyr Met Gln Lys 80 85 90 act ctg cgg caa tgt cag gaa cag agg cag tgtcac tgc agg cag gaa 439 Thr Leu Arg Gln Cys Gln Glu Gln Arg Gln Cys HisCys Arg Gln Glu 95 100 105 gcc acc aat gcc acc aga gtc atc cat gac aactat gat cag ctg gag 487 Ala Thr Asn Ala Thr Arg Val Ile His Asp Asn TyrAsp Gln Leu Glu 110 115 120 gtc cac gct gct gcc att aaa tcc ctg gga gagctc gac gtc ttt cta 535 Val His Ala Ala Ala Ile Lys Ser Leu Gly Glu LeuAsp Val Phe Leu 125 130 135 140 gcc tgg att aat aag aat cat gaa gta atgtcc tca gct tgatgacaag 584 Ala Trp Ile Asn Lys Asn His Glu Val Met SerSer Ala 145 150 gaacctgtat agtgatccag ggatgaacac cccctgtgcg gtttactgtgggagacagcc 644 caccttgaag gggaaggaga tggggaaggc cccttgcagc tgaaagtcccactggctggc 704 ctcaggctgt cttattccgc ttgaaaatag ccaaaaagtc tactgtggtatttgtaataa 764 actctatctg ctgaaagggc ctgcaggcca tcctgggagt aaagggctgccttcccatct 824 aatttattgt gaagtcatat agtccatgtc tgtgatgtga gccaagtgatatcctgtagt 884 acacattgta ctgagtggtt tttctgaata aattccatat tttacctatggaaaaaaaaa 944 aaaaaaaaaa aaaaaaaaaa aa 966 2 177 PRT Homo sapiens 2 MetLys Leu Gln Cys Val Ser Leu Trp Leu Leu Gly Thr Ile Leu Ile -20 -15 -10Leu Cys Ser Val Asp Asn His Gly Leu Arg Arg Cys Leu Ile Ser Thr -5 -1 15 Asp Met His His Ile Glu Glu Ser Phe Gln Glu Ile Lys Arg Ala Ile 10 1520 Gln Ala Lys Asp Thr Phe Pro Asn Val Thr Ile Leu Ser Thr Leu Glu 25 3035 40 Thr Leu Gln Ile Ile Lys Pro Leu Asp Val Cys Cys Val Thr Lys Asn 4550 55 Leu Leu Ala Phe Tyr Val Asp Arg Val Phe Lys Asp His Gln Glu Pro 6065 70 Asn Pro Lys Ile Leu Arg Lys Ile Ser Ser Ile Ala Asn Ser Phe Leu 7580 85 Tyr Met Gln Lys Thr Leu Arg Gln Cys Gln Glu Gln Arg Gln Cys His 9095 100 Cys Arg Gln Glu Ala Thr Asn Ala Thr Arg Val Ile His Asp Asn Tyr105 110 115 120 Asp Gln Leu Glu Val His Ala Ala Ala Ile Lys Ser Leu GlyGlu Leu 125 130 135 Asp Val Phe Leu Ala Trp Ile Asn Lys Asn His Glu ValMet Ser Ser 140 145 150 Ala 3 178 PRT Homo sapiens 3 Met His Ser Ser AlaLeu Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Ser ProGly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro Gly AsnLeu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45 Ser Arg Val LysThr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 Leu Leu Lys GluSer Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala LeuSer Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 Gln Ala GluAsn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110 Gly GluAsn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125 PheLeu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150155 160 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile165 170 175 Arg Asn 4 30 DNA Homo sapiens 4 ggcatgccat ggagttacagtgtgtttccc 30 5 24 DNA Homo sapiens 5 ggaagatcta gctgaggaca ttac 24 6 39DNA Homo sapiens 6 ggcgggatcc cgccatcatg aagttacagt gtgtttccc 39 7 36DNA Homo sapiens 7 cccaagcttg gtacctcatc aagctgagga cattac 36 8 36 DNAHomo sapiens 8 ggcgggatcc cgccatgaag ttacagtgtg tttccc 36 9 75 DNA Homosapiens 9 cccaagcttg gtacctcatc agaaagcgta gtctgggacg tcgtatgggtaagctgagga 60 cattacttca tgatt 75 10 39 DNA Homo sapiens 10 ggcgggatcccgccatcatg aagttacagt gtgtttccc 39 11 36 DNA Homo sapiens 11 cccaagcttggtacctcatc aagctgagga cattac 36 12 588 DNA Artificial cDNA 12 ataggtaaaatatggaattt attcagaaaa accactcagt acaatgtgta ctacaggata 60 tcacttggctcacatcacag acatggacta tatgacttca caataaatta gacgggaagg 120 cagccctttactcccaggat ggcctgcagg cctttcagca gatagagttt attacaaata 180 ccacagtagactttttggct attttcaagc ggaataagac agcctgaggc cagccagtgg 240 gactttcagctgccnggggc cttccccatc tccttcccct tcaaggtggg ctgtctccca 300 cagtaaaccgcacanggggt gttcatccct ggatcactat acaggttcct tgtcatcaag 360 ctgaggacattacttcatga ttcttattaa tccaggctag aaagacgtcg agctctccca 420 gggatttaatggcagcagcg tggacctcca gctgatcata gttgtcatgg atgactctgg 480 tggcattggtggcttctgnc tgcagtgaca atgcctctgt tctgacattg cgcagagttt 540 tctgcatgtagangaagagt tggcaatgng ctgattttct caagattt 588 13 514 DNA Artificial cDNA13 anaggtaaaa tatggaattt attcagaaaa accactcagt acaatgtgta ctacaggata 60tcacttggct cacatcacag acatggacta tatgacttca caataaatta gangggaagg 120cagcccttta ctcccaggat ggcctgcagg cctttcagca gatagagttt attacaaata 180ccacagtaga ctttttggct accttcaagc ggaataagac agcctgaggc cagccagtgg 240gactttcagc tgccnggggc cttccccatc tccttcccct tcaaggtggg ctgtctccca 300cagtaaaccg cacncggggt gttcatccct ggatcactat acaggttcct tgtcatcaag 360ctgaggacat tacttcatga ttcttattaa tccaggctag aaagacgtcg agctctccca 420gggatttaat ggcagcagcg tggaactcca gctgatcata gttgtcatgg atgacnctgg 480tggnttggtg gcttccggct gcagtgacat gcct 514 14 574 DNA Artificial cDNA 14accccaaaat cttgagaaaa atcagcagca ttgccgncgn gtnngggggt gngggggagg 60ngnngagnng nncnctntaa gagnccncnn aaangngttg ggaccaatgc caccagagtc 120atccatgaca actatgatca gctggaggtc cacgctgctg ccattaaatc cctgggagag 180ctcgacgtct ttctagcctg gattaatang aagnatggag gnnggtgngn ngcggggttg 240tgaggnngct gggangnggn gctgggtnng gagtngnngt tgccgcgnnt nggggagnna 300gtgcancctg aaggggaagg agatggggaa ggccccttgc agctgaaagt cccactggct 360ggcctcaggc tgtcttattc cgcttgaaaa tagccaaaaa gtctactgtg gtatttgtaa 420taaactctat ctgctgaaag ggcctgcagc aatcctggga gtaagggctg ccttcccanc 480taatttattg tgaagtcata tagtccatgt ctgtgatgtg agccaagtga tatctgtagt 540acacattgta ctgagtggtt ttctgaataa ttca 574 15 594 DNA Artificial cDNA 15caccccaaaa tcttgagaaa aatcagcagc attgccagnn cgnggcnggn ccgtgngcnn 60gnngnngnnn ncnggnngcn cncnttaaaa agccnnnnnn angggttcng ggnacccaat 120gccaccagag tcatccatga caactatgat cagctggagg tccacgctgc tgccattaaa 180tccctgggag agctcgacgt ctttctagcc tggattaata agaagcaggg ngggnggcgg 240ngngggcgtn ccgtgnncgg gnnaggggng ggncggtgng nnngcgcgnc gnggtggann 300nggtngcggn ggnngcgctg gggangtgnt nggaagggcc ctgcagctga aagtcccact 360ggctggcctc aggtgtctta ttccgcttga aaatagccaa aaagtctact gtggtatttg 420taataaactc tatctgctga aagggcctgc agcattcctg ggagtaaagg gctgccttcc 480catctaattt attgtgaagt catatagtcc atgtctgtga tgtgagccaa gtgatatcct 540gtagtacaca ttgtactgag tggtttttct gaataaattc atatttacct taaa 594 16 270DNA Artificial cDNA 16 gcactacttc cagaacacac aaggcctgat cttcgtggtggacagcaatg acagagagcg 60 tgtgaacgag gcccgtnagg agctcatgag gatgctggccgaggacgagn tccgggatgc 120 tgtcctcctg gtgttcgcca acaagcagga cctccccaacgncatgaatg cggccgagat 180 cacagacaag ctggggctgc actcactacg ccacaggaactggtacattc aggccacctn 240 cgncaccagc ggcgacgggc tctatgaagg 270 17 30 DNAHomo sapiens 17 acctgaggtc tgatggcaaa gtccaagaat 30

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about −24 toabout 153 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about −23 to about 153 in SEQ IDNO:2; (c) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 1 to about 153 in SEQ ID NO:2; (d) a nucleotidesequence encoding a polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97662; (e) a nucleotidesequence encoding the mature IL-19 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97662;and (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d) or (e).
 2. An isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a), (b), (c), (d) or (e)of claim 1 wherein said polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues.
 3. An isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a IL-19 polypeptide having an amino acidsequence in (a), (b), (c), (d), or (e) of claim
 1. 4. The isolatednucleic acid molecule of claim 3, which encodes an epitope-bearingportion of a IL-19 polypeptide comprising amino acid residues from about−5 to about 4 in SEQ ID NO:2.
 5. An isolated nucleic acid molecule,comprising a polynucleotide having a sequence selected from the groupconsisting of the nucleotide sequence of a fragment of the sequenceshown in SEQ ID NO:1 or the complement thereof, wherein said fragmentcomprises at least 50 contiguous nucleotides of SEQ ID NO:1, providedthat said fragment does not have a sequence starting: at nucleotide 9and ending at nucleotide 302 of SEQ ID NO:1 or any subfragment thereofor the complement thereof; or at nucleotide 438 and ending at nucleotide934 of SEQ ID NO:1 or any subfragment thereof or the complement thereof.6. A method for making a recombinant vector comprising inserting an isisolated nucleic acid molecule of claim 1 into a vector.
 7. Arecombinant vector produced by the method of claim
 6. 8. A method ofmaking a recombinant host cell comprising introducing the recombinantvector of claim 7 into a host cell.
 9. A recombinant host cell producedby the method of claim
 8. 10. A recombinant method for producing a IL-19polypeptide, comprising culturing the recombinant host cell of claim 9under conditions such that said polypeptide is expressed and recoveringsaid polypeptide.
 11. An isolated IL-19 polypeptide having an amino acidsequence at least 95% identical to a sequence selected from the groupconsisting of: (a) amino acids from about −24 to about 153 in SEQ IDNO:2; (b) amino acids from about −23 to about 153 in SEQ ID NO:2; (c)amino acids from about 1 to about 153 in SEQ ID NO:2; (d) the amino acidsequence of the IL-19 polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97662; (e) the aminoacid sequence of the mature IL-19 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97662;and (f) the amino acid sequence of an epitope-bearing portion of any oneof the polypeptides of (a), (b), (c), (d) or (e).
 12. An isolatedpolypeptide comprising an epitope-bearing portion of the IL-19 protein,wherein said portion is selected from the group consisting of: apolypeptide comprising amino acid residues from about −5 to about 4 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 64to about 82 in SEQ ID NO:2; and a polypeptide comprising amino acidresidues from about 115 to about 125 in SEQ ID NO:2.
 13. The isolatedpolypeptide of claim 11, which is produced or contained in a recombinanthost cell.
 14. The isolated polypeptide of claim 11, wherein saidrecombinant host cell is mammalian.
 15. An isolated nucleic acidmolecule comprising a polynucleotide encoding an IL-19 polypeptidewherein, except for at least one to fifty conservative amino acidsubstitution, said polypeptide has a sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a polypeptidecomprising amino acids from about −24 to about 153 in SEQ ID NO:2; (b) anucleotide sequence encoding a polypeptide comprising amino acids fromabout −23 to about 153 in SEQ ID NO:2; (c) a nucleotide sequenceencoding a polypeptide comprising amino acids from about 1 to about 153in SEQ ID NO:2; (d) a nucleotide sequence encoding a polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97662; (e) a nucleotide sequence encoding the mature IL-19polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97662; and (f) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d)or (e).
 16. An isolated IL-19 polypeptide wherein, except for at leastone to fifty conservative amino acid substitution, said polypeptide hasa sequence selected from the group consisting of: (a) amino acids fromabout −24 to about 153 in SEQ ID NO:2; (b) amino acids from about −23 toabout 153 in SEQ ID NO:2; (c) amino acids from about 1 to about 153 inSEQ ID NO:2; (d) the amino acid sequence of the IL-19 polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97662; (e) the amino acid sequence of the mature IL-19polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97662; and (f) the amino acid sequence ofan epitope-bearing portion of any one of the polypeptides of (a), (b),(c), (d) or (e).
 17. A method for treatment of an individual in need ofa decreased level of IFN-γ, TNF-α, or IL-6 activity comprisingadministering to said individual a composition comprising an isolatedpolypeptide of claim
 16. 18. A method for treatment of an individual inneed of an increase in IL-2 activity, comprising administering to saidindividual a composition comprising an antagonist of interleukin-19activity.